Protein biosynthesis involves the participation of a large number of macromolecules. Of particular interest are those protein factors which are involved in initiation and their interaction with ribosomes. Initiation factor-3 (IF-3) is crucial for this process by ensuring that a pool of free 30S ribosomal subunits is maintained and by stimulating the functional interaction of mRNA with the 30S subunit. The ribosomal protein components of the IF-3 binding site have been identified but to date this has not been accomplished for the 16S rRNA sequence which also comprises part of this site. In addition, little is currently known regarding the structure-function relationships of IF-3. Photochemical crosslinking of IF-3 to ribosomes has proven to be a useful technique for analysis of the IF-3 binding site. Employing this approach the 16S RNA sequences which participate in IF-3 binding will be identified. In addition, the ribosome binding site for IF-3 will be visualized directly by immunoelectron microscopy of IF-3-ribosomal subunit covalent complexes. Altered forms of IF-3 will be constructed by in vitro mutagenesis and the mutant IF-3s will be tested for functional activity. This should provide information on the composition of the active site of IF-3. The conservation of the IF-3 sequence in organisms other than E. coli at both the protein and DNA levels will be studied by heterologous hybridization and immunoblotting. In certain cases, the IF-3 homologues will be studied in greater detail with regard to both structure and function. These studies should facilitate identification of those segments of the IF-3 molecule which have been conserved and are presumably required for function. The biosynthesis of IF-3 and its regulation has not been examined in any detail. The gene encoding IF-3, infC, has been cloned in this laboratory and was shown to efficiently express IF-3. The putative promoter sequence(s) will be subcloned into a promoter fusion vector in order to study the activity and regulation of transcription which may originate from these sites. These fusion plasmids will also permit investigation of the nature of growth rate regulation of infC expression and whether this expression is subject to stringent control. The proposed experiments will result in the most comprehensive analysis of the ribosome binding site for IF-3 and a more complete description of the structure-function relationships of IF-3, as well as, clarify certain aspects of the regulation of IF-3 gene expression. Since IF-3 plays a central role in the initiation of protein synthesis, these studies will, in turn, provide for a more complete understanding of the molecular mechanism of this critical step in gene expression.